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Abstract

It was previously revealed that Wnt signaling is activated in mesothelioma cells. Although epidermal growth factor receptor (EGFR) is expressed in mesothelioma cells, EGFR‑tyrosine kinase inhibitors (TKIs) are not effective for mesothelioma treatment. However, in non‑small cell lung cancer, the blocking of Wnt signaling has been identified to enhance the anticancer effect of EGFR‑TKIs. To confirm the anticancer effect of blocking Wnt signaling in combination with EGFR‑TKI treatment in mesothelioma, the present study evaluated the effect of simultaneous suppression of human dishevelled‑3 (Dvl‑3) expression with Dvl‑3 small interfering RNA (siRNA) and of EGFR inhibition with gefitinib on mesothelioma cell viability. Mesothelioma cell lines with and without β‑catenin gene expression were transfected with Dvl‑3 siRNA and were cultured with gefitinib, and cell viability, colony formation and cell cycle analyses were performed. Dvl‑3 siRNA downregulated the expression of Dvl‑3 in mesothelioma cells. The combination of Dvl‑3 siRNA with gefitinib acted synergistically to induce concomitant suppression of cell viability and colony formation, suggesting that inhibition of Wnt signaling by downregulating Dvl‑3 with siRNA and inhibiting EGFR with gefitinib leads to significant antitumor effects.

Introduction

Malignant mesothelioma is an asbestos-associated
pleural malignancy that arises from serosal cells and presents a
poor prognosis. Statistical surveys conducted by the Japanese
Ministry of Health, Labor and Welfare reported that 1,410
fatalities were caused by mesothelioma in 2013 in Japan, double the
number of fatalities in 1999 (www.mhlw.go.jp/toukei/saikin/hw/jinkou/tokusyu/chuuhisyu15/dl/chuuhisyu.pdf).
Although therapies including surgery, chemotherapy and radiotherapy
have been adopted, the prognosis remains poor. Since the
combination of an anti-folate reagent, pemetrexed, and cisplatin
was demonstrated to prolong survival for longer than cisplatin
alone (1), this combination has been
frequently used, but its effects remain limited. Second-line
therapy for tumor recurrence has not been established. Newer
therapies based on an improved molecular understanding of
mesothelioma are therefore required.

Wnt signaling, which activates a canonical pathway
through β-catenin, is aberrantly activated in a wide range of
tumors (2). Numerous types of tumor,
including colon cancer, undergo aberrant activation of this
canonical pathway and Wnt may also activate non-canonical pathways
(2). Our previous study identified
that Wnt signaling is activated in mesothelioma cells, and that
blockade of Wnt signaling may be achieved with antibodies against
Wnt-1 or −2, Wnt-1 or −2 small interfering RNAs (siRNAs), or
dominant-negative dishevelled (Dvl), leading to suppressed
viability or tumorigenesis of mesothelioma cells in athymic mice
(3–7).
Notably, the mesothelioma H28 cell line exhibits Wnt signal
activity, which blocks apoptosis, without expression of β-catenin,
due to a homozygous deletion of the β-catenin gene and activation
of the aforementioned non-canonical pathways (8). Wnt signaling has been revealed to have a
crucial function in maintaining cancer stem cells, which are highly
resistant to chemotherapy (9).

The epidermal growth factor receptor (EGFR)-tyrosine
kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are
promising anticancer drugs for the treatment of patients with
non-small cell lung cancer (NSCLC) with a specific EGFR mutation
(10). EGFR has been revealed to be
expressed in 68% of paraffin-embedded mesothelioma specimens
(11). However, EGFR-TKIs alone are
not effective in the treatment of mesothelioma (12). In NSCLC, tankyrase, an upregulator of
the canonical Wnt signaling pathway, has been revealed to protect
lung cancer cells from EGFR inhibition (13). Inhibition of β-catenin with EGFR-TKIs
was reported to be able to enhance the anticancer effect or to
overcome the resistance to EGFR-TKIs (14–16). In
the present study, the effect of suppression of Wnt signaling with
Dvl-3 siRNA and of inhibition of EGFR with gefitinib on
mesothelioma cell viability were investigated.

Transfection with Dvl-3 siRNA

Dvl-3 siRNA (Stealth RNAi™) was prepared by
Invitrogen; Thermo Fisher Scientific, Inc. The sequence of the
siRNA and the methods of transfection have been described
previously (17). Briefly, cells were
seeded onto 35-mm dishes at 1×104 cells/dish, and were
transfected 24 h later with 4 pmol siRNA using 4 ml Lipofectamine™
2000 (Thermo Fisher Scientific, Inc.). Cells were then incubated
for 24 h at 37°C, washed once with PBS, then incubated with
RPMI-1640 medium containing 10% FBS at 37°C for >24 h for
western blot analysis and >14 days for colony formation assays
on 35-mm dishes, or seeded onto 96-well plates for cytotoxicity
assays.

Cytotoxicity assays

Cell viability was assessed using a modification of
the MTT assay using the Cell Counting Kit-8 (Dojindo Molecular
Technologies, Inc., Kumamoto, Japan) containing
2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium
and monosodium salt (WST-8) dye. Cells were seeded onto 96-well
plates at 5×103 cells/well 24 h after transfection with
siRNA. Cell viability was estimated at 24, 48, 72 and 96 h after
plating. WST-8 dye was added 2 h prior to the end of culture and
absorbance was measured at 450 nm using a Multiskan JX instrument
(Thermo Fisher Scientific, Inc.). Experiments were performed ≥6
times. At 24 h after plating, cells were exposed to 5, 10 or 30 µM
gefitinib (Sigma-Aldrich; Merck KGaA) or dimethylsulfoxide (DMSO)
as a control, and cell viability was estimated in the
aforementioned manner. Gefitinib was dissolved in DMSO and controls
for all experiments were created by adding equivalent volumes of
DMSO. Each drug concentration was added to three replicate wells
and each experiment was performed 4 times.

Colony formation assays

After 24 h of siRNA transfection, 400 cells were
spread onto 35-mm dishes with RPMI-1640 medium and 10% FBS, with 5
µM gefitinib or DMSO as a control. After 14 days, cells were
stained with 0.5% methylene blue for 24 h at room temperature and
colonies were counted visually. Colony assays were performed ≥4
times and results are reported as the mean.

Cell cycle analysis

After 24 h of siRNA transfection, 5 µM gefitinib or
DMSO was added to the medium. A further 24 h later, cells were
collected and a CycleTest PLUS DNA Reagent kit (BD Biosciences, San
Jose, CA, USA) was used according to the manufacturer's protocol.
Cell cycle analysis was determined using a flow cytometer (BD
FACSVerse™; version 1.0.3.2942; BD FACSuite software; BD
Biosciences).

Statistical analysis

Results are expressed as the mean ± standard
deviation. Data between two groups were compared using a two-tailed
unpaired Student's t-test. Analysis of variance (ANOVA), followed
by Dunnett's test, was used to compare multiple groups. For
cytotoxicity assays comparing concentrations of gefitinib, cells
treated with DMSO were used as a control and the viability of other
cells were compared using ANOVA followed by Dunnett's test.
Viability of cells transfected with control siRNA was compared with
that of those transfected with Dvl-3 siRNA using a two-sided
Student's t-test. P<0.05 was considered to indicate a
statistically significant difference. Statistical analysis was
performed using a commercial statistical software package (version
21; SPSS Statistics; IBM Corp., Armonk, NY, USA).

Results

Dvl-3 siRNA downregulates expression
of Dvl-3 in mesothelioma cells

Mesothelioma 211H, H2452 and H28 cells express Dvl-3
and EGFR. Whereas H28 cells expressed no β-catenin due to a
homozygous deletion of the β-catenin gene, 211H and H2452 cells
did. At 48 h after transfection with Dvl-3 siRNA, expression of
Dvl-3 was downregulated in 211H, H2452 and H28 cells (Fig. 1).

Downregulation of Dvl-3 and treatment
with gefitinib suppresses the viability of mesothelioma cells
synergistically

After 24 h of transfection with Dvl-3 siRNA or
control siRNA, gefitinib or DMSO was added to the medium. Following
transfection with Dvl-3 siRNA, the viability of H28 and 211H cells
was significantly suppressed compared with that of the cells
transfected with control siRNA, but the viability of H2452 cells
was not (Fig. 2). At 48 h after the
addition of gefitinib or DMSO, the viability of H28 cells
transfected with control siRNA was not suppressed in the presence
of 5 or 10 µM gefitinib; however, the viability of H28 cells
transfected with Dvl-3 siRNA was significantly suppressed following
treatment with these concentrations of gefitinib compared with
those treated with DMSO. The viability of 211H cells was
significantly suppressed following treatment with 30 µM gefitinib.
At 5, 10 or 30 µM gefitinib, the viability of 211H cells were
significantly suppressed synergistically with Dvl-3 siRNA
transfection compared with that of cells transfected with control
siRNA. In H2452 cells, 10 and 30 µM gefitinib significantly
suppressed the viability of cells transfected with Dvl-3 siRNA,
compared with that of those transfected with control siRNA.

Colony counts of 211H, H2452 and H28 cells were
significantly decreased following transfection with Dvl-3 siRNA,
and were further significantly decreased following treatment with 5
µM gefitinib, compared with respective controls (Fig. 3).

Downregulation of Dvl-3 and treatment
with gefitinib induces G1 population increase

After 48 h of transfection with Dvl-3 siRNA, the
population of 211H, H2452 and H28 cells in G1 phase tended to
increase (Fig. 4). At a further 24 h
after the addition of gefitinib to the medium, after 24 h of
transfection with Dvl-3 siRNA, the population of 211H, H2452 and
H28 cells in G1 phase tended to increase further, although these
results were not statistically significant (P>0.05; Fig. 4).

Discussion

The results of the present study indicate that
downregulation of Dvl-3 induced suppression of cell viability and
that the addition of the EGFR-TKI gefitinib acted synergistically,
resulting in colony formation with a tendency to persist in the G1
phase of the cell cycle. Of all pleural mesotheliomas, ~70% exhibit
high levels of EGFR expression (11).
Jänne et al (18) demonstrated
that 10 µM gefitinib suppressed the viability and colony formation
of mesothelioma cell lines in soft agarose. It has been
demonstrated that 10 µM gefitinib exceeds the effective dose in
NSCLC (13). In the present study,
inhibition of Dvl-3 enhanced inhibition of viability at 10 µM in
all three mesothelioma cell lines. In H28 cells, downregulation of
Dvl-3 suppressed cell viability, an effect which was enhanced 48 h
after treatment with 5 or 10 µM gefitinib. At 30 µM gefitinib, H28
cell viability was markedly decreased, but it was not affected by
downregulation of Dvl-3. Nutt et al (19) demonstrated that H28 cell viability was
completely suppressed 72 h after the addition of 30 µM gefitinib. A
concentration of 30 µM gefitinib is more toxic to H28 cells
compared with a concentration of 5 or 10 µM, and this toxicity may
not be associated with signaling pathways affected by the
downregulation of Dvl-3. The aim of colony formation assay
performed in the present study was to investigate the temporary
effect of suppression of Dvl-3 combined with treatment with an
EGFR-TKI on colony formation of mesothelioma cells. As colony
formation was suppressed in the present study, suppression of Dvl-3
may be associated with the initial expansion of cells. A limitation
of the present study is that the siRNA had no function after 14
days of transfection. It was confirmed that temporary transfection
of siRNA did not suppress Dvl-3 expression after 14 days (data not
shown). Future studies are required to examine colony formation
using short hairpin RNA in order to elucidate the effect on other
signaling pathways of continuous suppression of Dvl-3. In cell
cycle analysis, 5 µM gefitinib was used, and this dose did not
inhibit cell viability effectively 24 h after the addition.
Downregulation of Dvl-3 by siRNA usually induced G1 phase, which
tended to be enhanced by gefitinib, although these results were not
statistically significant. These results suggest that blockade of
the EGF signaling pathway by gefitinib or other EGFR-TKIs, and of
Wnt signaling by Dvl-3 suppression may be a useful combination for
the treatment of mesothelioma.

p-GSK3β (Ser9), which is the inactive
form of GSK3β and a regulator of Wnt signaling, and EGFR were
revealed to be negatively associated with survival of patients with
lung cancer, indicating that EGFR may phosphorylate GSK3β into
inactive p-GSK3β (20). GSK3β
participates in various critical cellular processes, one of which
is the formation of the β-catenin destruction complex (21). When Wnt signaling is not activated,
GSK3β is able to phosphorylate β-catenin, resulting in its
ubiquitination. Dvl family members inhibit activation of GSK3β and
degradation of β-catenin, which is translocated to the nucleus and
interacts with transcription factors, resulting in the expression
of target genes (21). The results of
the present study indicate that downregulation of Dvl-3 decreased
phosphorylation of GSK3β in 211H and H2452 cells. However, H28
cells without β-catenin expression exhibited a decrease in p-GSK3β
levels and total expression of GSK3β following downregulation of
Dvl-3. In 211H and H2452 cells, synergistic inhibition of cell
viability by Dvl-3 downregulation and gefitinib may be associated
with p-GSK3β. However, the precise function of GSK3β in EGFR and
Wnt signaling pathways in mesothelioma cells requires further
elucidation.

In NSCLC, Wnt signaling protects cells from
EGFR-TKIs via tankyrase or β-catenin (13–16). An
interaction between EGFR and Wnt signaling has been identified
(22,23). Numerous studies reviewed in Paul et
al (22) have demonstrated that
downregulation of β-catenin leads to a decreased expression of
EGFR, signal transducer and activator of transcription 3, cyclin
D1, matrix metalloproteinase (MMP)2, MMP9 and protein kinase B. In
mesothelioma cells, Wnt signaling and EGF signaling pathways may
support each other against cytotoxicity.

Dvl proteins relay Wnt signals from receptors to
downstream effectors, which activate either the canonical Wnt
pathway or the β-catenin-independent non-canonical pathway,
depending on the nuclear translocation of β-catenin (24). Previous studies have reported that the
suppression of Dvl inhibits the viability or tumorigenesis of
mesothelioma cells (3–7), and the cell viability of lung cancer
(4). Furthermore, our previous
studies demonstrated that mesothelioma cells express Dvl-3 and that
the inhibition of Dvl-3 suppressed mesothelioma cell viability,
including that of H28 cells, which do not express β-catenin
(8,17). This suggests that the activation of
Wnt signaling in mesothelioma cells may utilize the
β-catenin-independent non-canonical pathway. Zhao et al
(25) demonstrated that Dvl-3 induced
upregulation of p120-catenin, which is associated with cell
viability, invasion and metastasis of lung cancer. In lung cancer
cells, cytosolic transmembrane protein 88 was revealed to interact
with Dvl family members independently of β-catenin, which promoted
invasion and metastasis by activating p38-GSK3β-Snail signaling
(26).

The underlying molecular mechanism that led to these
results may be attributed to a reciprocal interaction of the EGFR
and Wnt signaling pathways. The function of Dvl-3 may differ among
cell lines utilizing the canonical and non-canonical pathways.
Notably, H28 cells do not express β-catenin, which is suspected to
be associated with a different signaling pathway from those
utilized by other mesothelioma cells. Although Dvl-3 is unable to
affect EGFR directly in H28 cells, downregulation of Dvl-3 may
inhibit other pathways to compensate for the negative effect
induced by inhibition of the EGFR pathway in H28 cells. However, in
order to fully understand these mechanisms, further studies are
required. Furthermore, the mechanism of cross-talk between these
pathways in mesothelioma cells remains to be elucidated.